Upon large-scale expression of therapeutic proteins, according to characteristics of host cells or target proteins, a target protein may vary in expression levels, water solubility, expression sites, modification, and the like. Thus, the most suitable expression system for a target protein must be selected to establish an effective production system.
Most therapeutic proteins are glycoprotiens where oligosaccharides are covalently bonded to asparagine residues as they pass through the endoplasmic reticulum (ER) and Golgi apparatus (Jenkins et al., Nat. Biotechnol., 14, 975-9, 1996). The structure and kind of sugar moieties greatly affect folding, biological activity and stability in serum of glycoprotiens. Thus, to date, for producing therapeutic recombinant glycoproteins having wild-type sugar moieties and therapeutic activity, the most commonly used approach is to use animal cell expression systems. However, there are drawbacks to animal cell culture systems, which include low yield, high cost due to expensive culture media, retroviral contamination, and a long period of time required for establishing stable cell lines. Thus, animal cell culture systems have limited applications in producing recombinant glycoproteins. In this regard, many attempts have been made to use, as an alternative to animal cell expression systems, yeast expression systems, which are eukaryotes and share the early steps of the N-linked glycosylation pathway of higher animal cells, to produce recombinant glycoproteins of medical importance.
Eukaryotes such as yeasts have advantages of rapidly producing high-yield proteins, utilizing sterilized and well-controlled production conditions, being easily genetically engineered, having no risk of infections by human or animal pathogens, and ensuring easy protein recovery. However, a complete type synthesized in yeasts has a different sugar moiety from that of target organisms such as mammalians, and thus may cause immune responses in animal cells. Also, this yeast-specific outer chain glycosylation of the high mannose type, also denoted hyperglycosylation, brings rise to heterogeneity of a recombinant protein product, which may make the protein purification complicated or difficult. Further, the specific activity of enzymes may be lowered due to the increased carbohydrate level (Bekkers et al., Biochem. Biophy. Acta. 1089, 345-351, 1991).
To solve the above problems, there is a need for glycotechnology which introduces into yeasts a glycosylation pathway of animal cells capable of producing glycoproteins having identical biological activity to those derived from mammalians.
When recombinant glycoproteins are expressed in traditional yeast, Saccharomyces cerevisiae, the addition of a series of 50 to 200 mannose residues to a core oligosaccharide, resulting in hypermannosylation, and the presence of α-1,3-linked terminal mannose recognizable as an antigen in the body were viewed as large constraints in employing the yeast as a host for glycoprotein production (Dean, Biochim. Biophys. Acta., 1426, 309-322, 1999; Ballou, Methods Enzymol., 185, 440-444, (1990)). By contrast, when recombinant glycoproteins are expressed in the methylotropic yeasts, Hansenula polymorpha and Pichia pastoris, they are expressed in a hypermannosylated form compared to natural forms, but the overall length of mannose outer chains is shorter than those expressed in S. cerevisiae (Kang et al., Yeast 14, 371-381, 1998; Kim et al., Glycobioloby, in press, 2004; Bretthauer and Castellino, Biotechnol. Appl. Biochem. 30, 193-200, 1999). In particular, since sugar chains synthesized in the methylotrophic yeasts, H. polymorpha and P. pastoris, do not contain the strongly immunogenic α-1,3-linked terminal mannose (Kim et al., Glycobioloby, in press, 2004; Montesino et al., Protein Expr. Purif. 14, 197-207, 1998), the methylotrophic yeasts are considered superior host systems to traditional yeast, S. cerevisiae, for the production of glycoproteins having therapeutic value in humans.
Many attempts were made in the glycotechnology field to develop hosts capable of producing therapeutic recombinant glycoproteins containing human compatible sugar chains using P. pastoris and S. cerevisiae (Chiba et al., J. Biol. Chem., 273, 26298-26304, 1998; Callewaert et al., FEBS Lett., 503, 173-178, 2001; Choi et al., Proc. Natl. Acad. Sci. USA, 100, 5022-5027, 2003; Hamilton et al., Science, 301, 1244-1246, 2003). For example, an attempt was made to produce a glycoprotein where an intermediate including the human mannose-type Man5GlcNAc2 N-glycan was attached using a recombinant S. cerevisiae obtained by further genetically manipulating a triple mutant yeast (och1Δmnn1Δmnn4Δ) to express mammalian α-1,2-mannosidase in the ER (Chiba et al., J. Biol. Chem., 273, 26298-26304, 1998). The triple mutant has disruption in three genes: OCH1 that plays a critical role in outer chain initiation (Nakanishi-Shindo et al., J. Biol. Chem. 268, 26338-26345, 1993; U.S. Pat. No. 5,705,616; U.S. Pat. No. 5,798,226); MNN1 that mediates addition of the immunogenic α-1,3-linked terminal mannose (Gopal and Ballou, Proc. Natl. Acad. Sci. USA 84, 8824, (1987); U.S. Pat. No. 5,135,854); and MNN4 that addes phosphates to a sugar chain (Jigami and Odani, Biochim. Biophys. Acta., 1426, 335-345, 1999). In addition, according to recent studies (Choi et al., Proc. Natl. Acad. Sci. USA, 100, 5022-5027, 2003; Hamilton et al., Science, 301, 1244-1246, 2003), host developments in P. pastoris were made to produce recombinant glycoproteins with the human complex-type N-glycan GlcNAc2Man3GlcNAc2 by introducing five different enzymes derived from eukaryotes into a secretory pathway in order to introduce the human glycosylation pathway into mutant strains (Japanese Pat. 07145005; Japanese Pat. 07150780; International Pat. Publication WO 0200856 A2; International Pat. Publication WO 0200879 A2) which have a disruption in the OCH1 gene mediating outer chain initiation. However, to date, from the viewpoint of glycotechnolgy, attempts have rarely been made to produce recombinant glycoproteins with human-type sugar chains in the methylotropic yeast H. polymorpha which is gaining popularity as a host for the expression of therapeutic recombinant proteins since it has been employed for producing hepatitis vaccines.
As described in Korean Pat. Application No. 2002-37717, the present inventors, before the present invention, cloned OCH1 gene playing a critical role in the outer chain synthesis of H. polymorpha, establishing a mutant strain (Hpoch1Δ) having a disrupted OCH1 gene, and developed a process for producing a recombinant glycoprotein with a sugar chain structure closer to a natural form by preventing hyperglycosylation using such a mutant. However, in the Hpoch1Δ mutant strain having a disruption in the OCH1 gene of H. polymorpha, outer chain glycosylation is still initiated by α-1,6-mannose linkage. Thus, there is a need for the finding of a gene coding for α-1,6-mannosyltransferase and prevention of the above human incompatible glycosylation pathway.